Self-calibrating gradient dilution in a constituent assay and gradient dilution apparatus performed in a thin film sample

ABSTRACT

A method and apparatus for measuring antibody titers in a thin film sample in an automated system which does not require multiple dilutions. The system provides a simple method for creating an in-situ dilution within a sample analysis chamber without the use of any precision fluid-handling components, and further, to use the same principles to provide a wide range of sample dilutions within the chamber so as to obviate the need for additional dilution steps when dealing with samples possibly containing wide ranges of analyte concentrations.

This application claims the benefit of U.S. Provisional ApplicationNos.: 61/041,784, filed Apr. 2, 2008; 61/041,791, filed Apr. 2, 2008;61/041,790, filed Apr. 2, 2008; 61/041,794, filed Apr. 2, 2008;61/041,797, filed Apr. 2, 2008; and 61/043,571, filed Apr. 9, 2008.

BACKGROUND OF THE INVENTION

1. Technical Field

This disclosure relates to methods and apparatus for measuring antibodytiters in an automated system which does not require multiple dilutions.The system provides a simple method for creating an in-situ dilutionwithin a sample analysis chamber without the use of any precisionfluid-handling components, and further, to use the same principles toprovide a wide range of sample dilutions within the chamber so as toobviate the need for additional dilution steps when dealing with samplespossibly containing wide ranges of analyte concentrations.

2. Background Information

In most assays it is necessary to provide an exact dilution of thesample to be analyzed so that the concentration of the analyte can bebrought into the useful range of the assay, and since this dilutionaffects the concentration of the analyte, the precision and accuracy ofthe test to a large extent depends upon the precision and accuracy ofthe dilution. One reason for this dilution is that immunoassays areaffected by a phenomenon known as the prozone effect. The term “prozone”as used in this disclosure shall refer to conditions of antibody excesswhere generally in precipitation or agglutination-based immunoassaysreactions are inhibited or prevented; the postpone, where conditions ofantigen excess in an immunoassay where agglutination or precipitationreactions are inhibited; and the “hook effect” where conditions ofantigen excess result in falsely low results. Conditions where theprozone effects occur can result in false negatives and falsely lowresults with catastrophic results to the patient.

Each assay combination has an empirically defined working range andassays must be performed with samples and reactants in the appropriatedilutions. This type of dilution has traditionally been accomplishedthrough the use of precision fluid-handling components or manualrepeating of the assay at higher dilutions of the antibody to see if thenegative is a true negative. Although these can be very accurate, theyrequire careful calibration and greatly add to the complexity ofautomated instrumentation. Additionally the range of analyte present inthe sample may exceed the dynamic range of the assay and may requirefurther dilution of the sample for accurate results.

Serologic assays, such as for antibodies to infectious diseasepathogens, are important in that they tell of either existing immunitydue to immunization or to previous or current exposure, depending on theclass of immunoglobulin present, to the infectious agent. Similarly,they may be used to detect auto-immunity and the like. There are anumber of assay types performed, including agglutination,complement-fixation, precipitation, etc. One almost universal feature ofsuch tests is the need to dilute the sample a number of times in orderto detect the point where the antibodies are no longer effective tocause a positive test. This is referred to as the “titer”, the titerbeing the highest dilution of the patient's serum or plasma that yieldsdetectable agglutination or measured reaction with the test antigen.This, in effect, requires the performance of many separate tests toarrive at the result. Another problem with such assays is that theend-points are sometimes difficult to determine, thus adding asignificant error to the titer determination. Automation can increasethe test efficiency and accuracy, but performing the dilutions by aninstrument is very difficult and time consuming including the need tofirst define the desired dilution which can vary from test to test andthe multiple dilution steps are very complex.

It would be desirable to provide a method and apparatus for measuringantibody titers in an automated system which does not require multipledilutions and that removes the risk of false negatives due to theprozone effect.

SUMMARY OF THE INVENTION

According to an aspect of the present invention, a sensible marker isused to permit the measurement of the concentration of the reactantsadded to the in vitro chamber in the area of the reaction beinganalyzed. A sensible marker in this disclosure means a dye or detectablesubstance that does not interfere with the reaction being analyzed andthat diffuses at a rate close the reactants to which it is added.Sensible markers may be a dye or dyes that can be measured by opticalmeans such as absorption or fluorescent emission. The sensible marker ishomogeneously present either being in solution or colloidal suspensionwith at least one of two or more liquids to be subsequently added to,and allowed to mix in, the thin analysis chamber being used.

Since the height of the chamber is less than 100 microns (100μ), andpreferable less than 20 microns (20μ), and the lateral dimensions of thechamber are preferably several centimeters, the greater than 1,000 folddifference in the vertical and horizontal dimensions will result inequilibrium being reached in the vertical dimension extremely rapidlywhile the equilibrium in the lateral dimension will take hundreds tothousands of times longer. If the entire image of the reaction chamberimaged or scanned and discrete small areas of the image or scan areanalyzed, where the lateral aspects of the discrete analysis areas arein the range of 1 to 3 times the height or the chamber, the volume beingsubjected to the analysis will be in approximate equilibrium. Areastaken at millimeter distances or greater, lateral to the first area willhave different equilibrium conditions. The signal from the admixedsensible marker is measured before and after subsequent mixing ordiffusion with the additional reactants, to permit calculation of finalmeasured sensible marker concentration, reflects the relative dilutionof the components. In cases where there are more than two liquidspresent in a chamber, more than one sensible marker that is able to bedistinguished from the other sensible markers may be employed, eachadded to one of the added components, to enable the calculation ofrelative proportions of each of the components. If the initialconcentration of the constituents of the components is known, therelative concentrations may be used to calculate the absoluteconcentration of the added components in mass per unit volume. Thus therelative concentrations of added reactants in any small analyzed areamay be treated as a virtual discrete reaction vessel or chamber whoseconcentrations of added reagents is calculable and the results for thebound over free or agglutination or other signal employed in theimmunoassay being performed may be measured and plotted as the signalobtained per calculated dilution of sample or standard per concentrationof added antibody or added antigen.

It is therefore an object of this invention to provide a method andapparatus wherein mixing and diffusion are used to create aconcentration gradient between two or more miscible liquids in a thinfilm sample in a chamber so that the equilibrium in the thin dimensionof the chamber is very rapid and concentration differences in the longaxis of the chamber do not reach equilibrium during the time of theassay, and the final relative inter-dilution being measured by therelative concentration of a sensible marker which does not participatein any of the desired chemical reactions and whose properties are suchthat it allows its accurate measurement at any point in the reactionchamber.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic plan view of a chamber which is used in theperformance of the method of this invention;

FIG. 2 is a cross sectional view of the chamber of FIG. 1;

FIG. 3 is an enlarged cross sectional view of the chamber of FIG. 1showing a pumping of the solution in the chamber by deflection of thetop surface of the chamber to facilitate the establishment of differentconcentrations throughout the lateral aspects of the chamber:

FIG. 4 is a plan view of the chamber of FIG. 1 after the pumping stephas been completed;

FIG. 5 shows a trace of fluorescent emission readings from the chamberof FIG. 1 as taken along line a-a of FIG. 4 where a sensible marker is afluorescent dye;

FIG. 6 is a plan view of the chamber of FIG. 1 wherein the chamber hasinternal baffles which will cause sample mixing when the sample is firstintroduced into the chamber whereby physical manipulation of the sampleis not needed;

FIG. 7 is a schematic plan view similar to FIG. 1, but with a relativelysmall sample in the chamber;

FIG. 8 is a plan view similar to FIG. 7 but showing the sample aftermixing;

FIG. 9 is a schematic plan view of the chamber of FIG. 1 but showing theresult of adding three liquids to the chamber;

FIG. 10 is a schematic cross sectional view of a test chamber formed inaccordance with this invention;

FIG. 11 is a view of the test chamber similar to FIG. 10, showingagglutination of particles after adding a test sample to the chamber;

FIG. 12 is a cross sectional view similar to FIG. 10 showing antibodiespresent in the test chamber before the test sample is added to thechamber;

FIG. 13 is a view similar to FIG. 11 showing agglutination of particlesafter adding a test sample to the chamber;

FIG. 14A is a compound plan view of a test chamber which shows thepresence of agglutinated particles in the sample; and

FIG. 14B is a graph of the agglutinated particles in the sample takenfrom a scan along line a-a, and showing the cut off location T of theabsence of particle agglutination in the sample.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 is a schematic top view of a chamber 1, in this instance asquare, whose cross-section is shown in FIG. 2. The chamber is comprisedof relatively thin top and bottom plates, at least one of which must betransparent. Into the chamber are introduced two or more liquids, onebeing the sample 3 to be analyzed and the other being the reagent 4required for the analysis. At least one of these liquids has a dissolvedmarker which may be fluorescent, such as fluorescence, or an absorbentdye, such as phenol red, or the like. The marker must be such that itdoes not chemically interfere with the desired analytical signal norshould the marker signal be affected by any signal or reaction productsof the analysis in a manner which cannot be compensated for.

In the instance shown, liquid 4 is the analyzing reagent which containsa fluorescent marker, and liquid 3 is the sample to be analyzed. If theliquids are introduced into the chamber in equal amounts, in thedirections indicated, they will meet approximately at region 5. FIG. 3,which is also an enlarged cross-sectional view of the chamber,demonstrates how the liquids may be partially mixed. If one of thechamber surfaces is “pumped” up and down, mixing of the liquids willoccur, approximately along line 6, resulting in the dilution gradientshown in FIG. 4, which is a top view of the chamber.

After a suitable period of mixing, the chamber is allowed to stand for avariable period in order to allow vertical diffusion to complete themixing of the liquids within a given vertical segment. At this point,the fluids in regions 7 and 8 are still completely undiluted andrepresent the native state of the liquids before mixing. If fluorescencereadings from the marker are then taken along line a-a, the result canbe seen in FIG. 5, which is a cross-sectional view of the chamber alongline a-a, with a superimposed graph showing the fluorescence of thechamber at each relative position and a second graph showing the opticalabsorbance from the analyte.

Since signal level 9 represents that from the undiluted markeredreagent, and signal level 10 represents the background level of thesample, the chamber region corresponding to signal level 11 contains asample which has been diluted exactly by half. Thus, the analyteconcentration inferred from the signal of the desired reaction may bemultiplied by two to obtain the exact concentration. If, in thisinstance, it is known that the analyte signal is too high due to thepresence of too much analyte in the mixture in that region, one needonly find a region with a marker signal equivalent to that of region 12,which is a greater dilution, and then multiply the analyte absorbanceresult accordingly.

Similarly, in conditions where the prozone effect is present, theinstrument reports the highest analyte result obtained after taking alldilutions into account and also reports that this calculation has beenperformed.

The sample may be mixed by other means then “pumping” the chamber. Forexample, FIG. 6 is a schematic top view of a chamber with baffles 13which serve to cause sample mixing when the liquids are introduced asshown.

It is not necessary for some portion of either the sample or the reagentto remain undiluted. For example, in FIG. 7, which is another schematictop view of a chamber with a relatively small sample 14, where in thiscase the sample is the liquid containing the marker, and a large reagentarea 15 which does not contain the marker. Prior to mixing, referencereadings are taken over regions 16 and 17, and after mixing (FIG. 8),there is no remaining undiluted sample, but the original referencevalues can be used for the same calculations as described above. Thisparticular instance, where a marker is uniformly mixed with the sample,is particularly suited for instances where a relatively high dilutionratio is required.

All of the instances shown show the formation of a dilution gradient,but this may not always be necessary. In cases where a single,approximate dilution will suffice, the sample and markered reagent (ormarkered sample) can be mixed to uniformity and a reading taken from anysuitable region, again using the marker concentration to calculate thefinal actual dilution.

In the above instances, it was assumed that the thickness of the chamberwas uniform, but this is not absolutely required. It would be acceptableto a chamber having a thickness at the point of measurement that isknown or can be determined from other means; e.g., the absolute readingposition in the case of a chamber of defined geometric shape, or athickness that can measured by means independent of the marker, such asinterferometry or by the systems described in U.S. Pat. Nos. 6,127,184,6,723,290 and 6,929,953, which patents are hereby incorporated byreference in their entirety.

The chamber thickness must be sufficiently small that convection cellsdo not develop, and also small enough that complete vertical mixing bydiffusion can occur in a reasonable period of time. In the preferredembodiment, the chamber is less than 1 mm thick, and preferably lessthan 200μ. The area of the chamber is largely irrelevant, but for mostapplications an area of about 4 cm² is adequate.

In instances where the chamber must be incubated for a prolonged timefollowing mixing in order for a reaction to proceed, the gradient maytend to decrease due to diffusion beyond desired bounds. In these cases,a viscosity increasing agent, such as dextran, polyoxyethylene or thelike, or by an agent which can form at least a partial gel, such asgelatin or agar, can be used to delay further diffusion.

An additional particularly important application of this invention isthe means by which it can be used to provide a simultaneous standardcurve and analytical dilution. Standard curves are frequently used tocalibrate a given analysis, where known standards of varyingconcentrations are analyzed to generate a response curve of analyticalsignal vs. sample concentration. When the sample containing the unknownconcentration of analyte is then measured, the analytical signal iscompared to the standard curve to give the concentration of the analytein the sample. This necessitates multiple analyses, and if the reactionis not repeatable over time, this may require a repetition of thisprocess with every analytical run. A similar situation exists with theuse of control material, which is, in effect, standards of knownconcentration, which are analyzed along with the sample in a batch inorder to ensure that the analysis is working properly. Both of thesesituations can be avoided by a particular use of the describedinvention.

FIG. 9 shows a sample cell 18 where three liquids are introduced, thesample containing the unknown concentration of analyte, the reagentcontaining the marker, and a standard of appropriate concentration.Baffles 19 may be used to prevent complete mixing of the constituents.When the chamber has equilibrated as previously described, readingsalong line 21 are used to generate a standard curve, using thepreviously described method, and readings along line 20 are used to findthe appropriate sample dilution for the analysis. Thus, a simultaneousstandard curve and sample analysis can be performed in the same reactionchamber, which ensures that the reaction conditions for the sample andstandard are identical. More than one sample could be run in a singlechamber by altering the geometry, as long as the appropriate mixingoccurs. What is being measured is light per pixel of the area scanned.

An agglutination assay is performed in the test chamber as described,with the following features added to affect a serologic assay.

FIG. 10 is a schematic cross-sectional view of a test chamber having atleast one transparent surface 101 of the general construction describedabove. To one surface of the chamber are adhered particles 102 whosesurfaces express or contain the antigen 103 to which the target antibodyis directed. The particles may be artificial, such as latex,latex-styrene, styrene, polycarbonate, or the like, with antigen bondedto the surface by any of several means well known to the art, or theymay be natural, such as pollen, bacteria, yeast, mold or fungus. Theparticles must be of such a size so as to enable the determination thatparticle agglutination has occurred, and are most preferably in a sizerange of 0.2μ to 20μ. The particles are adhered to, and preferablycovered by, a soluble coat 104, which may be comprised of sugars, suchas trehalose, which preserves the activity of the antigen 103.

When a liquid sample 105 containing the antibodies to be detected 106 isadded to the chamber, the soluble coat 104 dissolves, releasing theparticles 102 and exposing their adhered antigen 103 to the antibody106. As shown in FIG. 11, which shows the chamber of FIG. 10 some timeafter the sample has been added, the antibody 106 in the sample, ifpresent in sufficient quantity, will cause the particles to agglutinateto form at least pairs of particles 107, or if present in higherconcentration, to form larger clumps 108. It is readily apparent thatinspection of the chamber by an automated instrument can detect thepresence of clumping of the particles by any number of image-processingalgorithms well known to the art.

In the example given, the antibody 106 was presumed to be polyvalent,such as Ig-M, which is the antibody formed in the early stages of aresponse to an infection. If the immune response is longer lasting,however, Ig-G antibody will be present, which is not polyvalent and isless effective in causing the clumping. To effect a better clumping inthat case, the soluble layer 104 should contain a polyvalent anti-Fcantibody active to link the Fc fragments of the non-polyvalent antibody110 to be detected. Thus, when layer 104 dissolves, the anti-Fc antibody109 is released and binds the antibodies 110, in effect, creating a formof polyvalent antibody 110 which can clump the particles 102 as shown inFIGS. 12 and 13.

FIG. 14A is a schematic top view of a chamber combining the features ofthe above-cited disclosure and the instant disclosure, and a graphdepicting the presence of aggregated particles versus the position alongline a-a. Sample 112, admixed with a marker as previously described, anda diluent 113 is introduced into a chamber in a manner so as to allowthe formation of a gradient dilution. After a suitable incubation periodwhich will depend upon the nature of the antigen and antibody beingdetected, the chamber is scanned along line a-a and the region T islocated, as seen in FIG. 14B, which represents the position whereagglutination or clumping no longer occurs. The reciprocal of thedilution of the sample at this point, as determined by the relativeconcentration of the marker in this area, is equal to the titer of theantibody. For example, if the marker concentration is 0.2 compared tothat in the original sample area 112, the titer is 5.

It should be noted that other immunological reactions besidesagglutination or clumping can be detected, such as precipitation, wherethe antigen and antibodies form a visible complex instead of clumpingparticles. It should also be noted that the means described in thepresent invention may also be employed in other types of immunoassays,including those where the method of analysis includes the virtualsubtraction off bound from free, the subject of the copending U.S.Provisional Patent Application No. 61/041,784, filed Apr. 2, 2008 andDocket No. 7564-0035-1, filed presently herewith. In the latter case,with the present invention there is no need to avoid prozone effects,but the present invention can be used to optimize the working range onthe assay and may be performed without deviation from the specificationscontained in the present disclosure.

Although the invention has been shown and described with respect tospecific detailed embodiments thereof, it will be understood by thoseskilled in the art that various changes in form and detail thereof maybe made without departing from the spirit and the scope of theinvention.

1. A method for determining the relative dilution of a solutioncontaining a ligand which is directed against a target analyte, and aliquid sample containing the target analyte to be analyzed, which targetanalyte sample is miscible with said ligand solution, at least one ofsaid solution and said sample containing a detectable marker, saidmethod comprising the steps of: a) placing an effective amount of saidsolution in one area of a planar reaction chamber having a thin throughthickness dimension; b) placing an effective amount of said targetanalyte sample in an adjacent area of said planar chamber; c) allowingor causing said solution and said sample to admix with each other untilan admixture gradient is created in a solution-sample mixture in saidchamber; d) electronically imaging or scanning said diffusionconcentration gradient in said chamber from a first location containingthe sensible-undiluted-markered-mixed-ligand solution and a secondlocation containing the undiluted target analyte sample; and e)calculating a relative concentration or dilution of each of saidsolution and sample from an imaged area by measuring the concentrationof any marker in said mixture in said imaged area.
 2. The method ofclaim 1 wherein said solution contains a detectable sensible marker. 3.The method of claim 1 wherein said liquid sample contains a detectablesensible marker.
 4. The method of claim 1 wherein both said solution andsaid liquid sample contain detectable sensible markers and wherein saidsensible markers produce different detectable signals.
 5. The method ofclaim 1 wherein said allowing or causing admixing step is performed bypumping said solution and said sample in said chamber.
 6. The method ofclaim 1 wherein said allowing or causing admixing step is performed bybaffling flow of at least one of said solution and liquid sample intosaid chamber.
 7. The method of claim 1 wherein said chamber is less than1 mm in thickness.
 8. The method of claim 6 wherein said chamber is lessthan 200μ in thickness.
 9. The method of claim 1 comprising the furtherstep of adding a viscosity increasing agent which can form at least apartial gel in said sample so as to allow the sample to be incubated foran extended period of time following said admixing step in order for adelayed reaction to proceed.
 10. The method of claim 9 wherein a prozoneeffect is avoided.
 11. The method of claim 1 wherein the risk of falsenegatives due to a prozone effect is removed by expanding the range ofthe immunoassay to assure more accurate immunoassay results.
 12. Themethod of claim 1 wherein a standard calibration curve is performed inthe same chamber as the sample analysis.
 13. A method for determiningthe relative dilution of a solution containing a ligand which isdirected against a target analyte, and a liquid sample containing thetarget analyte to be analyzed, which target analyte sample is misciblewith said ligand solution, at least one of said solution and said samplecontaining a detectable sensible marker, said method comprising thesteps of: a) placing a first amount of said solution in a first area ofa planar reaction chamber having a thin through thickness dimension; b)placing a second amount of said target analyte sample in a second areaof the chamber, which second area is adjacent the first area of saidplanar chamber, and which second amount is less than the first amount;c) allowing or causing said solution and said sample to admix with eachother until an even solution-sample mixture is created in said chamber;d) electronically imaging or scanning said resultant solution-samplemixture in said chamber to determine sensible marker signal; and e)electronically calculating a relative concentration or dilution of eachof said solution and sample from any imaged area by measuring theconcentration of any marker in said mixture in said imaged area.
 14. Themethod of claim 13 wherein said solution contains a detectable sensiblemarker.
 15. The method of claim 13 wherein said liquid sample contains adetectable sensible marker.
 16. The method of claim 13 wherein both saidsolution and said liquid sample contain detectable sensible markers andwherein said sensible markers produce different detectable signals. 17.The method of claim 13 wherein said allowing or causing admixing step isperformed by pumping said solution and said sample in said chamber. 18.The method of claim 13 wherein said allowing or causing admixing step isperformed by baffling flow of at least one of said solution and liquidsample into said chamber.
 19. The method of claim 13 wherein saidchamber is less than 1 mm in thickness.
 20. The method of claim 19wherein said chamber is less than 200μ in thickness.